Organ fibrosis and subsequent organ failure are estimated to account for at least one third of deaths worldwide. Pulmonary fibrosis is a relatively rare form of fibrosis but there are presently no effective treatments; mean survival rates following diagnosis are 3 to 5 years. In fibrosis of the lung, as in other organs, the primary cell type that drives disease is the myofibroblast. This cell type is derived by direct conversion of other cell types in response to cell signaling factors such as TGF?. Calpain, a cysteine protease, has been implicated in the development of fibrosis but its role has not been fully explored. In genetic studies, mice that lack calpain activity do not develop pulmonary fibrosis when exposed to the toxin, bleomycin. We extended these results by demonstrating that fibroblasts isolated from these mice, unlike their wild-type counterparts, do not form myofibroblasts in response to TGF? treatment. Phelix Therapeutics has developed a series of potent calpain inhibitors that show promising results in initial attempts to block myofibroblast generation in a cell-based model. We propose to further develop this model by employing quantitative measures of myofibroblast formation. We will use this model to evaluate the ability of calpain inhibitors to prevent myofibroblast formation from a variety of lung cell types. Finally, our best inhibitors will be tested in pharmacokinetic and ADME studies and optimized to improve their properties in vivo for subsequent use in efficacy studies in a mouse model of pulmonary fibrosis.!